Hydrophobic interaction chromatography (HIC) is a method for separating proteins based on the strength of their relative hydrophobic interactions with a hydrophobic adsorbent. Hydrophobicity is generally defined as the repulsion between a non-polar compound and a polar environment, such as water. Hydrophobic “interactions” are essentially the tendency of a polar environment to exclude non-polar (i.e., hydrophobic) compounds from the polar environment and force aggregation of the hydrophobic amongst themselves. The phenomenon of hydrophobic interactions is applied to the separation of proteins by using an aqueous salt solution to force a hydrophobic protein in a sample to aggregate with or bind adsorptively to hydrophobic functional groups (the adsorbent) affixed to a solid support. The adsorbed proteins are released from the adsorbent by eluting with decreasing salt concentrations which reverse the environment promoting the hydrophobic interactions, leading to loss of hydrophobic interactions between the proteins and the support and release of the protein from the support in order of increasing hydrophobicity (with the least hydrophobic proteins being released first).
Recombinant proteins typically contain a variety of impurities that need to be removed before the product is pharmaceutically acceptable. Some of these impurities may include host cell proteins (HCPs) from the host cell system in which they are expressed. For a CHO system, these impurities are referred to as CHO Host Cell Proteins (CHOP). In addition to these impurities, the protein as expressed during cell culture may also contain variant forms of the product protein, for example, a misfolded form of the target protein. Other impurities may be added to the product stream or generated as a result of the purification process, such as higher molecular weight aggregates of the protein or leached Protein A. These impurities have a wide range of retentions on different modes of chromatography and removal of such a broad spectrum of impurities is difficult, typically requiring multiple steps involving different modes of chromatography.
HIC may be utilized to separate proteins using two different approaches. In the first HIC approach, referred to as the “bind and elute” mode, the mixture containing the target protein is contacted with the hydrophobic adsorbent under conditions where the target protein binds to the adsorbent, while contaminants (or as much of the contaminants as possible) do not bind and flow through. In the “bind and elute” mode, the target protein may be recovered by applying to the adsorbent/protein complex a salt concentration applied in a gradual or step-wise reduced gradient, to selectively release the various bound proteins and contaminants and collecting discreet fractions until the fraction containing the more purified protein is obtained. In a process where a target protein is bound to the column (while allowing contaminants to flow through), adsorbents having greater hydrophobicity are typically used to bind a broader range of proteins which will be collected in a specific fraction release at a specific salt concentration in the course of applying the salt gradient. In the second HIC approach, referred to as the “flow-through” mode, the mixture containing the target protein is contacted with the hydrophobic adsorbent under conditions where the contaminants (or as much of the contaminants as possible) bind to the adsorbent, while the target protein (and as few contaminants as possible) does not bind and flows through. In this mode, the use of less hydrophobic adsorbents, such as those having lower molecular weight alkyl groups, are preferred, since a lower binding capacity is needed for conditions under which the target protein does not bind. As one would expect, however, use of HIC in the flow-through mode has been of limited usefulness because the conditions needed to allow the target protein to flow through inherently result in lower binding capacities, leading to early elimination of the target protein, or elimination of the target protein along with contaminants.
While several different modalities of chromatography can be employed to remove a particular class of impurities, very few chromatographic steps are capable of removing all these impurities from a product. Thus, there is need for a purification process that can be employed generically for removal of these impurities from a recombinant protein.